Quantitative gene expression profiling

ABSTRACT

Described herein are approaches to the measurement of gene expression for chosen genes in a biological sample. The methods permit the quantitation of target nucleic acids, e.g., DNAs or RNAs in a nucleic acid sample, both singly and in a multiplex format that permits the determination of levels (e.g., expression levels or copy numbers) for two or more target nucleic acids in a single reaction.

FIELD OF THE INVENTION

The invention relates to methods and compositions for the measurement ofnucleic acids. More particularly, the invention relates to methods andcompositions for measuring levels of DNA and/or RNA in biologicalsamples.

BACKGROUND OF THE INVENTION

The introduction of genomics has been instrumental in accelerating thepace of drug discovery. The genomic technologies have proved their valuein finding novel drug targets. Further improvement in this area willprovide more efficient tools resulting in faster and more cost efficientdevelopment of potential drugs.

The drug discovery process includes several steps: the identification ofa potential biochemical target associated with disease, screening foractive compounds and further chemical design, preclinical tests, andfinally clinical trials. The efficiency of this process is still farfrom perfect: it is estimated that about 75% of money spent in the R&Dprocess went to fund failed projects. Moreover, the later in the productdevelopment a failure occurs, the bigger are losses associated with thisproject. Thus it is important to eliminate early in the process futurefailures in order to reduce costs of the whole drug development process.Thus, the quality of the original molecular target becomes a decisivefactor for cost-effective drug development.

One approach that promises to impact on the process of targetidentification and validation is transcription profiling. This methodcompares expression of genes in a specific situation: for example,between disease and normal cells, between control and drug-treated cellsor between cells responding to treatment and those resistant to it. Theinformation generated by this approach can directly identify specificgenes to be targeted by a therapy, and, importantly, reveals biochemicalpathways involved in disease and treatment. In brief, it not onlyprovides biochemical targets, but at the same time, a way to assess thequality of these targets. Moreover, in combination with cell-basedscreening, transcription profiling is positioned to dramatically changethe field of drug discovery. Historically, screening for a potentialdrug was successfully performed using phenotypic change as a marker infunctional cellular system. For example, growth of tumor cells inculture was monitored to identify anticancer drugs. Similarly, bacterialviability was used in assays aimed at identifying antibiotic compounds.Such screens were typically conducted without prior knowledge of thetargeted biochemical pathway. In fact, the identified effectivecompounds often revealed such pathways and pointed out the truemolecular target, enabling subsequent rational design of the nextgenerations of drugs.

Modern tools of transcription profiling can be used to design novelscreening methods that will utilize gene expression in place ofphenotypic changes to assess effectiveness of a drug. For example, somemethods are described in U.S. Pat. Nos. 5,262,311; 5,665,547; 5,599,672;5,580,726; 6,045,988 and 5,994,076, as well as Luehrsen et al. (1997,Biotechniques, 22:168-74; Liang and Pardee (1998, Mol Biotechnol.10:261-7). Such approaches will be invaluable, for example, for drugdiscovery in the field of central nervous system (CNS) disorders such asdementia, mild cognitive impairment, depression, etc., where phenotypicscreening is inapplicable, but where a transcription profile can beestablished and linked to particular disorders. Once again, theidentified effective compounds will likely reveal the underlyingmolecular processes. This approach can also be instrumental for thedevelopment of improved versions of existent drugs, which act at severalbiochemical targets at the same time to generate a desiredpharmacological effect. In such case the change in the transcriptionalresponse may be a better marker for drug action than selection based onoptimization of binding to multiple targets.

In addition to uses in drug development, transcriptional profiling andother measurements of nucleic acid presence or abundance can be used fordiagnosis, for example, where expression, overexpression or lack ofexpression of a particular gene or set of genes correlates with a givendisease state or predisposition. Similarly, where copy number(amplification, deletion or disruption) of a gene sequence at thechromosomal level correlates with a disease or disease predisposition,determination of DNA copy number in an individual or in a tissue or celltype can predict or diagnose that disease.

Common methods of transcription profiling are based on technology usingDNA microarrays, for example, as reviewed in Greenberg, 2001 Neurology57:755-61; Wu, 2001, J Pathol. 195:53-65; Dhiman et al., 2001, Vaccine20:22-30; Bier et al., 2001 Fresenius J Anal Chem. 371:151-6; Mills etal., 2001, Nat Cell Biol. 3:E175-8; and as described in U.S. Pat. Nos.5,593,839; 5,837,832; 5,856,101; 6,203,989; 6,271,957; and 6,287,778.The DNA microarray approach performs simultaneous comparison of theexpression of several thousand genes in a given sample by assessinghybridization of the labeled polynucleotide samples, obtained by reversetranscription of mRNAs, to the DNA molecules attached to the surface ofthe test array.

The microarray approach screens the pool of genes presented in themicroarray. The current printing methods allows placement of10,000-15,000 genes on a single chip, which is essentially a number ofgenes expressed in a particular cell type. Given the diversity of celltypes, it requires development of specific arrays for specific celltypes. Microarrays tend to provide qualitative, rather than quantitativeresults.

The number of transcripts in a tissue sample is even higher than in acellular sample and can exceed the capacity of a microarray.

Exogenous control involves the use of an artificially introduced nucleicacid molecule that is added, either to the extraction step or to the PCRstep, in a known concentration. The concept of adding an exogenousnucleic acid at a known concentration in order to act as an internalstandard for quantitation was introduced by Chelly et al. (1988) Nature333: 858-860, which is specifically incorporated herein by reference.Therefore, utilizing a control fragment that is amplified with the sameprimers as the target sequence more accurately reflects target sequenceamplification efficiency relative to the internal standard (see, forexample, WO 93/02215; WO 92/11273.; U.S. Pat. Nos. 5,213,961 and5,219,727, all of which are incorporated herein by reference). Similarstrategies have proven effective for quantitative measurement of nucleicacids utilizing isothermal amplification reactions such as NASBA(Kievits et al., 1991, J. Virol. Methods 35: 273-86) or SDA (Walker,1994, Nucleic Acids Res. 22: 2670-7).

Capillary electrophoresis has been used to quantitatively detect geneexpression. Rajevic at el. (2001, Pflugers Arch. 442(6 Suppl 1):R190-2)discloses a method for detecting differential expression of oncogenes byusing seven pairs of primers for detecting the differences in expressionof a number of oncogenes simultaneously. Sense primers were 5′end-labelled with a fluorescent dye. Multiplex fluorescent RT-PCRresults were analyzed by capillary electrophoresis on ABI-PRISM 310Genetic Analyzer. Borson et al. (1998, Biotechniques 25:130-7) describesa strategy for dependable quantitation of low-abundance mRNA transcriptsbased on quantitative competitive reverse transcription PCR (QC-RT-PCR)coupled to capillary electrophoresis (CE) for rapid separation anddetection of products. George et al., (1997, J Chromatogr B Biomed SciAppl 695:93-102) describes the application of a capillaryelectrophoresis system (ABI 310) to the identification of fluorescentdifferential display generated EST patterns. Odin et al. (1999, JChromatogr B Biomed Sci Appl 734:47-53) describes an automated capillarygel electrophoresis with multicolor detection for separation andquantification of PCR-amplified cDNA.

Omori et al. (2000, Genomics 67:140-5) measures and compares the amountof commercially purchased α-globin mRNA by competitive PCR in twoindependently reverse transcribed cDNA samples using oligo(dT) or oligo(dU) primers. The oligo(dT) or oligo (dU) primers share a 3′ oligo(dT)or oligo (dU) sequence and a 5′ common sequence. In addition theoligo(dT) or oligo (dU) primer for each sample also contains a unique 29nucleotide sequence between the 3′ oligo(dT) or oligo (dU) sequence andthe 5′ common sequence. After the synthesis of first strand cDNA, PCR isperformed to amplify the cDNA using a gene-specific primer and a primercomplementary to the common sequence which is labeled with a uniquelabel. The amplified PCR products are then analyzed by spotting onto adetection plate of a fluorescence scanner.

SUMMARY OF THE INVENTION

Disclosed herein are methods for determining the amount of one or anumber of target polynucleotides, e.g., DNAs or RNAs, in a givenbiological sample. The methods described herein permit the determinationof, for example, gene expression levels of one or more target genesequences, in a high throughput manner suited to the development andcomparison of gene expression profiles in biological samples. Geneexpression profiles may be thought of as a snapshot of the expressionstate of a set of target genes in, for example, a given tissue ororganism. The comparison of gene expression profiles can providevaluable information, for example, with regard to mechanisms of diseaseand the activity of known or potential drug candidates.

In one aspect, the invention relates to methods of estimating ordetermining the level of a target nucleic acid, e.g., a DNA or RNA in anucleic acid sample, the method comprising: for a given target nucleicacid, selecting a pair of amplification primers that will generate atarget amplicon of known length upon amplification of the target, e.g.,by PCR or RT-PCR. A set of at least two competitor nucleic acids (e.g.,DNA or RNA molecules) is generated, where the competitors yield productsof differing lengths but similar amplification efficiencies relative tothe target nucleic acid when amplified using the same pair ofamplification primers. An amplification reaction is performed in which asample to be analyzed for target nucleic acid level is mixed with knownand differing concentrations of the at least two competitor nucleicacids, followed by separation and detection of the amplified products.The set of competitor nucleic acids provides an internal reference forthe determination of target nucleic acid amount in the original sample.This approach is readily adapted to measure multiple target nucleicacids in a single sample in a single run, which permits the generationof an amplification profile for the selected target gene sequences in agiven sample.

Definitions:

As used herein, the term “amplicon” refers to an amplification productfrom a nucleic acid amplification reaction. The term generally refers toan anticipated, specific amplification product of known size, generatedusing a given set of amplification primers.

As used herein, the term “reverse transcript” refers to a DNA complementof an RNA strand generated by an RNA-dependent DNA polymerase activity.

As used herein, the term “competitor nucleic acid” or “nucleic acidcompetitor” refers to a nucleic acid template of known length andcomposition which can be amplified using a pair of oligonucleotideprimers selected for the amplification of a target nucleic acid. Incertain embodiments, the competitor nucleic acid can be an RNA molecule,in which case it can be referred to as a “competitor RNA” or an “RNAcompetitor.” In other embodiments, the competitor nucleic acid can be aDNA molecule, in which case it can be referred to as a “competitor DNA”or a “DNA competitor.” A “competitor nucleic acid” (whether DNA or RNA)is longer or shorter than the target nucleic acid, e.g., by a known,distinguishable length, e.g., the length of an internal insertion ordeletion in the target nucleic acid, respectively. The internalinsertion or deletion should be from 1 to 20 nucleotides or bases,preferably 5 to 20 nucleotides or bases, or 5 to 10 nucleotides orbases. The difference in length of the target and competitor ampliconswill be from 1 to 20 nucleotides in length, preferably 5 to 20 or 5 to10 nucleotides in length. Inserted sequence will preferably notintroduce the capacity for stable secondary structure not present in thetarget sequence. Software for predicting nucleic acid secondarystructure is well known in the art. A “competitor nucleic acid” willhave an amplification efficiency that is similar to that of the targetnucleic acid when using a selected pair of amplification primers.

As used herein, the term “similar efficiency” when applied to nucleicacid amplification, means that the threshold cycle (Ct) for thedetection of target and competitor nucleic acid amplification productsgenerated using the same set of primers and equal amounts of target andcompetitor template is the same. It is possible to calculate Ct to afraction of a cycle. However, the Ct for one amplicon is “the same” asthe Ct for another amplicon when the whole cycle numbers are thesame—i.e., Ct's of 2.0, 2.3 and 2.6 are “the same” as the term is usedherein. As used herein, “Ct” is the PCR cycle at which at which signalintensity of PCR product reaches a threshold value of 10 standarddeviations of background value of signal intensity for an amplifiedproduct. Signal intensity in this context refers to fluorescent signalfrom amplification product incorporating fluorescent label (either bylabeled primer or labeled nucleotide incorporation), measured followingcapillary electrophoresis of amplified products present in sampleswithdrawn from a cycling reaction at a plurality of cycle points.Another measure of amplification efficiency is to measure the amount ofamplification product (e.g., by fluorescence integrity or labelincorporation) at successive cycles, calculating efficiency using theformula E=(P_(n+1)−P_(n))/(P_(n)−P_(n−1)), where P=the amount ofamplification product at cycle n. Amplification efficiency is “similar”if the difference in efficiency between target and competitor nucleicacid is less than 0.2 in absolute value.

In the methods described herein, efficiency is “similar” if theefficiency of amplification of target and competitor nucleic acid is“similar” by either of these criteria, and preferably, by both.

As used herein, reference to “separating” or the “separation of” nucleicacids in a sample refers to a method of nucleic acid separation capableof resolving nucleic acid fragments that differ in size by 10 bases orless (or, alternatively, by 10 base pairs or less, e.g., wherenon-denaturing conditions are employed). Preferred resolution forseparation techniques employed in the methods described herein includesresolution of nucleic acids differing by 5 nucleotides or less(alternatively, 5 base pairs or less), up to and including resolution ofnucleic acids differing by only one nucleotide (or one base pair).

As used herein, reference to a “size distinguishable by capillaryelectrophoresis” means a difference of at least one nucleotide (or basepair), but preferably at least 5 nucleotides (or base pairs) or more, upto and including 10 nucleotides (or base pairs) or more.

As used herein, the term “sample” refers to a biological material whichis isolated from its natural environment and contains a polynucleotide.A “sample” according to the invention may be tissue or cell extract orit may contain purified or isolated polynucleotide.

As used herein, the term “amplified product” refers to polynucleotideswhich are copies of a particular polynucleotide, produced in anamplification reaction. An “amplified product,” according to theinvention, may be DNA or RNA, and it may be double-stranded orsingle-stranded.

As used herein, the term “amplification” or “amplification reaction”refers to a reaction for generating a copy of a particularpolynucleotide sequence or increasing the copy number or amount of aparticular polynucleotide sequence. For example, polynucleotideamplification may be a process using a polymerase and a pair ofoligonucleotide primers for producing any particular polynucleotidesequence, i.e., the whole or a portion of a target polynucleotidesequence, in an amount which is greater than that initially present.Amplification may be accomplished by the in vitro methods of thepolymerase chain reaction (PCR). See generally, PCR Technology:Principles and Applications for DNA Amplification (H. A. Erlich, Ed.)Freeman Press, NY, N.Y. (1992); PCR Protocols: A Guide to Methods andApplications (Innis et al., Eds.) Academic Press, San Diego, Calif.(1990); Mattila et al., Nucleic Acids Res. 19: 4967 (1991); Eckert etal., PCR Methods and Applications 1: 17 (1991); PCR (McPherson et al.Ed.), IRL Press, Oxford; and U.S. Pat. Nos. 4,683,202 and 4,683,195,each of which is incorporated by reference in its entirety. Otheramplification methods include, but are not limited to: (a) ligase chainreaction (LCR) (see Wu and Wallace, Genomies 4: 560 (1989) and Landegrenet al., Science 241: 1077 (1988)); (b) transcription amplification (Kwohet al., Proc. Natl. Acad. Sci. USA 86: 1173 (1989)); (c) self-sustainedsequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990)); and (d) nucleic acid based sequence amplification (NABSA)(see, Sooknanan, R. and Malek, L., Bio Technology 13: 563-65 (1995)),each of which is incorporated by reference in its entirety.

As used herein, the term “aliquot” refers to a sample volume taken froman amplification reaction mixture. The volume of an aliquot can vary,but will generally be constant within a given experimental run. Analiquot will be less than the volume of the entire reaction mixture.Where there are X aliquots to be withdrawn during an amplificationregimen, the volume of an aliquot will be less than or equal to 1/Xtimes the reaction volume.

As used herein, the term “dispense” means dispense, transfer, withdraw,extrude or remove.

As used herein, the phrase “dispensing an aliquot from the reactionmixture at plural stages” refers to the withdrawal of an aliquot atleast twice, and preferably at least 3, 4, 5, 10, 15, 20, 30 or moretimes during an amplification reaction. A “stage” will refer to a pointat or after a given number of cycles, or, where the amplificationregimen is non-cyclic, will refer to a selected time at or after theinitiation of the reaction.

As used herein, a “target polynucleotide” (including, e.g., a target RNAor target DNA) is a polynucleotide to be analyzed. A targetpolynucleotide may be isolated or amplified before being analyzed usingmethods of the present invention. For example, the target polynucleotidemay be a sequence that lies between the hybridization regions of twomembers of a pair of oligonucleotide primers which are used to amplifyit. A target polynucleotide may be RNA or DNA (including, e.g., cDNA). Atarget polynucleotide sequence generally exists as part of a larger“template” sequence; however, in some cases, a target sequence and thetemplate are the same.

As used herein, an “oligonucleotide primer” refers to a polynucleotidemolecule (i.e., DNA or RNA) capable of annealing to a polynucleotidetemplate and providing a 3′ end to produce an extension product which iscomplementary to the polynucleotide template. The conditions forinitiation and extension usually include the presence of four differentdeoxyribonucleoside triphosphates (dNTPs) and a polymerization-inducingagent such as a DNA polymerase or reverse transcriptase activity, in asuitable buffer (“buffer” includes substituents which are cofactors, orwhich affect pH, ionic strength, etc.) and at a suitable temperature.The primer as described herein may be single- or double-stranded. Theprimer is preferably single-stranded for maximum efficiency inamplification. “Primers” useful in the methods described herein are lessthan or equal to 100 nucleotides in length, e.g., less than or equal to90, or 80, or 70, or 60, or 50, or 40, or 30, or 20, or 15, butpreferably longer than 10 nucleotides in length.

As used herein, “label” or “detectable label” refers to any moiety ormolecule which can be used to provide a detectable (preferablyquantifiable) signal. A “labeled nucleotide” (e.g., a dNTP), or “labeledpolynucleotide”, is one linked to a detectable label. The term “linked”encompasses covalently and non-covalently bonded, e.g., by hydrogen,ionic, or Van der Waals bonds. Such bonds may be formed between at leasttwo of the same or different atoms or ions as a result of redistributionof electron densities of those atoms or ions. Labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, massspectrometry, binding affinity, hybridization radiofrequency,nanocrystals and the like. A nucleotide useful in the methods describedherein can be labeled so that the amplified product may incorporate thelabeled nucleotide and becomes detectable. A fluorescent dye is apreferred label according to the present invention. Suitable fluorescentdyes include fluorochromes such as Cy5, Cy3, rhodamine and derivatives(such as Texas Red), fluorescein and derivatives (such as 5-bromomethylfluorescein), Lucifer Yellow, IAEDANS, 7-Me₂N-coumarin-4-acetate,7-OH-4-CH₃-coumarin-3-acetate, 7-NH₂-4-CH₃-coumarin-3-acetate (AMCA),monobromobimane, pyrene trisulfonates, such as Cascade Blue, andmonobromorimethyl-ammoniobimane (see for example, DeLuca,Immunofluorescence Analysis, in Antibody As a Tool, Marchalonis, et al.,eds., John Wiley & Sons, Ltd., (1982), which is incorporated herein byreference).

It is intended that the term “labeled nucleotide”, as used herein, alsoencompasses a synthetic or biochemically derived nucleotide analog thatis intrinsically fluorescent, e.g., as described in U.S. Pat. Nos.6,268,132 and 5,763,167, Hawkins et al. (1995, Nucleic Acids Research,23: 2872-2880), Seela et al. (2000, Helvetica Chimica Acta, 83:910-927), Wierzchowski et al. (1996, Biochimica et Biophysica Acta,1290: 9-17), Virta et al. (2003, Nucleosides, Nucleotides & NucleicAcids, 22: 85-98), the entirety of each is hereby incorporated byreference. By “intrinsically fluorescent”, it is meant that thenucleotide analog is spectrally unique and distinct from the commonlyoccurring conventional nucleosides in their capacities for selectiveexcitation and emission under physiological conditions. For theintrinsically fluorescent nucleotides, the fluorescence typically occursat wavelengths in the near ultraviolet through the visible wavelengths.Preferably, fluorescence will occur at wavelengths between 250 nm and700 nm and most preferably in the visible wavelengths between 250 nm and500 nm.

The term “detectable label” or “label” include a molecule or moietycapable of generating a detectable signal, either by itself or throughthe interaction with another label. The “label” may be a member of asignal generating system, and thus can generate a detectable signal incontext with other members of the signal generating system, e.g., abiotin-avidin signal generation system, or a donor-acceptor pair forfluorescent resonance energy transfer (FRET) (Stryer et al., 1978, Ann.Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300).

The term “nucleotide,” as used herein, refers to a phosphate ester of anucleoside, e.g., mono, di, tri, and tetraphosphate esters, wherein themost common site of esterification is the hydroxyl group attached to theC-5 position of the pentose (or equivalent position of a non-pentose“sugar moiety”). The term “nucleotide” includes both a conventionalnucleotide and a non-conventional nucleotide which includes, but is notlimited to, phosphorothioate, phosphite, ring atom modified derivatives,and the like, e.g., an intrinsically fluorescent nucleotide.

As used herein, the term “conventional nucleotide” refers to one of the“naturally occurring” deoxynucleotides (dNTPs), including dATP, dTTP,dCTP, dGTP, dUTP, and dITP.

As used herein, the term “non-conventional nucleotide” refers to anucleotide which is not a naturally occurring nucleotide. The term“naturally occurring” refers to a nucleotide that exists in naturewithout human intervention. In contradistinction, the term“non-conventional nucleotide” refers to a nucleotide that exists onlywith human intervention. A “non-conventional nucleotide” may include anucleotide in which the pentose sugar and/or one or more of thephosphate esters is replaced with a respective analog. Exemplary pentosesugar analogs are those previously described in conjunction withnucleoside analogs. Exemplary phosphate ester analogs include, but arenot limited to, alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates,phosphoroanilidates, phosphoroamidates, boronophosphates, etc.,including any associated counterions, if present. A non-conventionalnucleotide may show a preference of base pairing with another artificialnucleotide over a conventional nucleotide (e.g., as described in Ohtsukiet al. 2001, Proc. Natl. Acad. Sci., 98: 4922-4925, hereby incorporatedby reference). The base pairing ability may be measured by the T7transcription assay as described in Ohtsuki et al. (supra). Othernon-limiting examples of “artificial nucleotides” may be found in Lutzet al. (1998) Bioorg. Med. Chem. Lett., 8: 1149-1152); Voegel and Benner(1996) Helv. Chim. Acta 76, 1863-1880; Horlacher et al. (1995) Proc.Natl. Acad. Sci., 92: 6329-6333; Switzer et al. (1993), Biochemistry 32:10489-10496; Tor and Dervan (1993) J. Am. Chem. Soc. 115: 4461-4467;Piccirilli et al. (1991) Biochemistry 30: 10350-10356; Switzer et al.(1989) J. Am. Chem. Soc. 111: 8322-8323, all of which herebyincorporated by reference. An “non-conventional nucleotide” may also bea degenerate nucleotide or an intrinsically fluorescent nucleotide.

As used herein, the term “degenerate nucleotide” denotes a nucleotidewhich may be any of dA, dG, dC, and dT; or may be able to base-pair withat least two bases of dA, dG, dC, and dT. An unlimiting list ofdegenerate nucleotide which base-pairs with at least two bases of dA,dG, dC, and dT include: Inosine, 5-nitropyrole, 5-nitroindole,hypoxanthine, 6H,8H,4-dihydropyrimido[4,5c][1,2]oxacin-7-one (P),2-amino-6-methoxyaminopurine, dPTP and 8-oxo-dGTP.

As used herein, the term “opposite orientation”, when referring toprimers, means that one primer comprises a nucleotide sequencecomplementary to the sense strand of a target polynucleotide template,and another primer comprises a nucleotide sequence complementary to theantisense strand of the same target polynucleotide template. Primerswith an opposite orientation may generate a PCR amplified product frommatched polynucleotide template to which they complement. Two primerswith opposite orientation may be referred to as a reverse primer and aforward primer.

As used herein, the term “same orientation”, means that primers comprisenucleotide sequences complementary to the same strand of a targetpolynucleotide template. Primers with same orientation will not generatea PCR amplified product from matched polynucleotide template to whichthey complement.

As used herein, a “polynucleotide” generally refers to anypolyribonucleotide or poly-deoxyribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. “Polynucleotides” include, withoutlimitation, single- and double-stranded polynucleotides. The term“polynucleotides” as it is used herein embraces chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including for example, simple and complex cells. A polynucleotideuseful for the present invention may be an isolated or purifiedpolynucleotide or it may be an amplified polynucleotide in anamplification reaction.

As used herein, “isolated” or “purified” when used in reference to apolynucleotide means that a naturally occurring sequence has beenremoved from its normal cellular environment or is synthesized in anon-natural environment (e.g., artificially synthesized). Thus, an“isolated” or “purified” sequence may be in a cell-free solution orplaced in a different cellular environment. The term “purified” does notimply that the sequence is the only nucleotide present, but that it isessentially free (about 90-95%, up to 99-100% pure) of non-nucleotide orpolynucleotide material naturally associated with it.

As used herein, the term “cDNA” refers to complementary or copypolynucleotide produced from an RNA template by the action ofRNA-dependent DNA polymerase activity (e.g., reverse transcriptase).

As used herein, “complementary” refers to the ability of a single strandof a polynucleotide (or portion thereof) to hybridize to ananti-parallel polynucleotide strand (or portion thereof) by contiguousbase-pairing between the nucleotides (that is not interrupted by anyunpaired nucleotides) of the anti-parallel polynucleotide singlestrands, thereby forming a double-stranded polynucleotide between thecomplementary strands. A first polynucleotide is said to be “completelycomplementary” to a second polynucleotide strand if each and everynucleotide of the first polynucleotide forms base-paring withnucleotides within the complementary region of the secondpolynucleotide. A first polynucleotide is not completely complementary(i.e., partially complementary) to the second polynucleotide if onenucleotide in the first polynucleotide does not base pair with thecorresponding nucleotide in the second polynucleotide. The degree ofcomplementarity between polynucleotide strands has significant effectson the efficiency and strength of annealing or hybridization betweenpolynucleotide strands. This is of particular importance inamplification reactions, which depend upon binding betweenpolynucleotide strands.

An oligonucleotide primer is “complementary” to a target polynucleotideif at least 50% (preferably, 60%, more preferably 70%, 80%, still morepreferably 90% or more) nucleotides of the primer form base-pairs withnucleotides on the target polynucleotide.

As used herein, the term “analyzing,” when used in the context of anamplification reaction, refers to a qualitative (i.e., presence orabsence, size detection, or identity etc.) or quantitative (i.e.,amount) determination of a target polynucleotide, which may be visual orautomated assessments based upon the magnitude (strength) or number ofsignals generated by the label. The “amount” (e.g., measured in μg, μmolor copy number) of a polynucleotide may be measured by methods wellknown in the art (e.g., by UV absorption, by comparing band intensity ona gel with a reference of known length and amount), for example, asdescribed in Basic Methods in Molecular Biology, (1986, Davis et al.,Elsevier, N.Y.); and Current Protocols in Molecular Biology (1997,Ausubel et al., John Weley & Sons, Inc.). One way of measuring theamount of a polynucleotide in the present invention is to measure thefluorescence intensity emitted by such polynucleotide, and compare itwith the fluorescence intensity emitted by a reference polynucleotide,i.e., a polynucleotide with a known amount.

The practice of the methods described herein will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology and recombinant DNA techniques, which are within the skillof the art. Such techniques are explained fully in the literature. See,e.g., Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: ALaboratory Manual, Second Edition; Oligonucleotide Synthesis (M. J.Gait, ed., 1984); Polynucleotide Hybridization (B. D. Harnes & S. J.Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal,1984); and a series, Methods in Enzymology (Academic Press, Inc.); ShortProtocols In Molecular Biology, (Ausubel et al., ed., 1995). Thepractice of the present invention may also involve techniques andcompositions as disclosed in U.S. Pat. Nos. 5,965,409; 5,665,547;5,262,311; 5,599,672; 5,580,726; 6,045,998; 5,994,076; 5,962,211;6,217,731; 6,001,230; 5,963,456; 5,246,577; 5,126,025; 5,364,521;4,985,129; as well as in U.S. patent application Ser. Nos. 10/113,034;10/387,286; 10/719,185; 10/600,201; 10/752,123 and 10/719,746. Allpatents, patent applications, and publications mentioned herein, bothsupra and infra, are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic of one embodiment of a quantitative nucleicacid measurement as described herein.

DESCRIPTION

Described herein are approaches to the measurement of gene expressionfor chosen genes in a biological sample. The methods permit thequantitation of target nucleic acids, e.g., DNAs or RNAs in a nucleicacid sample, both singly and in a multiplex format that permits thedetermination of levels (e.g., expression levels or copy numbers) fortwo or more target nucleic acids in a single reaction.

The methods described herein use internal standards generated throughthe use of known differing concentrations of exogenously addedcompetitor nucleic acids that generate amplification products of knownsizes that differ from each other and from the size of the targetnucleic acid(s). Size separation by, for example, capillaryelectrophoresis, coupled with detection by, for example, fluorescencedetection, generates a standard curve from the abundance of theamplification products corresponding to the competitor nucleic acids.The standard curve permits the determination of the target nucleic acidconcentration(s) in the original sample.

In one aspect, then, there is described a method of estimating the levelof a target nucleic acid in a nucleic acid sample. That method comprisesthe following steps. First, for a given target nucleic acid, a pair ofamplification primers is selected that will generate a target ampliconof a known length following reverse-transcription (for RNA target) andamplification (e.g., PCR amplification, for both RNA and DNA targets)using that pair of primers. Considerations for primer design are wellknown to those of skill in the art; however, among the more criticalaspects are specificity, i.e., the primers chosen should amplify onlythe desired target molecule under at least one set of amplificationconditions, and compatibility with additional primers that may beemployed in a reaction, e.g., where multiplex analyses are to beperformed. The length and nucleotide content (e.g., the G+C content) ofthe oligonucleotide primer is instrumental in determining thespecificity and hybridization characteristics (e.g., meltingtemperature) of the primer. Further considerations for oligonucleotideprimer selection or design are known to those of skill in the art and/ordescribed herein below.

Next, a set of at least two competitor nucleic acids is created. Thecompetitor nucleic acids share the same primer binding sequences (ortheir complements) for the selected amplification primers as the targetnucleic acid, but differ in the length of the amplicon that will begenerated using the same set of amplification primers used to amplifythe target sequence. It is important that the at least two competitornucleic acids have similar amplification efficiencies (as the term isdefined herein) relative to each other and to the target nucleic acidswhen the selected pair of amplification primers is used to generate anamplification product from each. In the set of at least two competitornucleic acids, it is preferred that one competitor generates a longeramplicon using the same primers, and another generates a shorteramplicon. (As discussed herein below, additional longer or shortercompetitors can also be included in differing amounts, e.g., to modifythe resolution of the assay.) In other embodiments, each of the at leasttwo competitor nucleic acids can generate a longer amplicon than thatgenerated from the target nucleic acid. It should be understood that inthis instance, each of the competitors should generate amplicons ofdiffering known lengths relative to each other and to the targetamplicon. In other embodiments, each of the at least two competitornucleic acids can generate a shorter amplicon than that generated fromthe target nucleic acid—here again, the competitor amplicons must differby known lengths from each other and from the target amplicon. Methodsof generating nucleic acids for use in the methods described herein arewell known in the art, e.g., PCR (for DNA competitors) or in vitrotranscription from plasmid or other isolated template DNA (for RNAcompetitors), or chemical synthesis. Methods for PCR, in vitrotranscription and for the generation of templates that differ in lengthfrom a given DNA template are well known to those of skill in the artand/or described herein below.

The difference in size of the competitor nucleic acid amplicons shouldbe a difference that can be detected by a method capable ofdistinguishing nuclei acids differing in size by 10 nucleotides/basepairs or less, and preferably by 5 nucleotides/base pairs or less, oreven by as little as 1 nucleotide or base pair. A well-suited method is,for example, capillary electrophoresis. Conditions under which capillaryelectrophoresis permits the detection of length differences of as littleas one nucleotide are well known. While differences of as little as onenucleotide are intended to be encompassed within the methods describedherein, it is preferable that the difference between competitors andtarget be at least 5 nucleotides, in order to better resolve theresulting amplicons from the target amplicon upon separation by, forexample, capillary electrophoresis. Differences greater than 5nucleotides are also contemplated, e.g., 10, 20, 30, 40 or 50nucleotides. However, the difference should not be so great as to renderthe efficiency of amplification significantly different (i.e., resultingin a difference in amplification efficiency E of greater than 0.2 inabsolute value, where E=(P_(n+1)−P_(n))/(P_(n)−P_(n−)) (where P_(n) isthe amount of PCR product at cycle n) with respect to the efficiency ofthe target amplicon or the at least one other competitor amplicon(s).Factors affecting the efficiency of amplification are well known tothose of skill in the art and include, for example, T_(m) of theprimers, the length of the amplicon, nucleotide composition of theamplicon, potential for secondary structure in the target or in theprimers, and the presence of, for example, modified nucleotides in thereaction. The measurement of amplification efficiency and factorsaffecting it are known to those of skill in the art and/or describedherein below.

One straightforward approach to generating competitor nucleic acidsinvolves the internal insertion or deletion of sequences from thesequence of the target amplicon. This approach maximizes thesimilarities between the competitor nucleic acids and the target nucleicacids, which in turn makes it more likely that amplificationefficiencies will be similar. Thus, one would perform site-directedmutagenesis on a cloned or amplified copy of the sequence (e.g., acloned cDNA) corresponding to the target nucleic acid, to either add ordelete nucleotide sequence sufficient to change the size of the amplicongenerated when the selected pair of primers is used for amplification.Of course, it should be clear that one would not mutate the sequencesbound by the selected primer pair. Site-directed mutagenesis can beperformed by any of a number of methods well known in the art.

It can be useful to generate sets of three, four or more competitornucleic acids. Having additional competitors can either expand or morenarrowly define the range of quantitative determination within a givenassay. That is, when first and second competitors are used at, forexample, a range of concentrations between 10 and 10,000 molecules in areaction, concentrations of target nucleic acid between 10 and 10,000molecules in a given volume of the original sample can be determinedfrom the standard curve generated by the competitors. While thisdetermination can be quite accurate, a narrower range of competitorconcentrations, e.g., 10 to 500 or 1,000 molecules can increase theaccuracy. Similarly, where a first estimate is to be made, the range canbe broader, e.g, 10 to 50,000 molecules, with later reactions run atnarrower concentrations if desired to more accurately determine thetarget nucleic acid concentration. It can be advantageous to includethree, four or more competitor nucleic acids for a given target nucleicacid at different concentrations in a given reaction. One of skill inthe art will recognize that as the concentration of competitors goes up,there may need to be an adjustment in the amount of amplificationprimers or other parameters for the amplification reaction.

Once a pair of amplification primers is selected and a set of competitornucleic acids is generated, target nucleic acids in a sample can bequantitated by combining a test nucleic acid sample with the set of atleast two competitor nucleic acid molecules, reverse transcribing thetarget and competitor nucleic acids and amplifying the target andcompetitor sequences using the pair of amplification primers. In analternative approach, competitor nucleic acids can be added to a sampleprior to extraction of nucleic acid from the test sample. In thisinstance, target and competitor nucleic acids will be co-isolated.

In order to be most accurate, the competitors should be added to thesample such that at least one is added at a known concentration belowthat of the target nucleic acid and at least one is added at a knownconcentration above that of the target nucleic acid. The knownconcentrations of competitor nucleic acids should differ by at least anorder of magnitude (i.e., 10-fold), but can advantageously differ byseveral orders of magnitude, e.g., at 100-fold, 1,000 fold or more. Ifthe amount of target nucleic acid expected is completely unknown, it canbe advantageous to perform one or more preliminary experiments usingdifferent ranges of competitors, in order to identify an anticipatedrange of concentrations for the given target. Alternatively, one oranother of a number of less accurate quantitative amplificationapproaches can be employed to garner a rough estimate of theconcentration to expect. Such methods are known in the art and use, forexample, titration in a series of parallel reactions against a singlereference template.

Reverse transcription is used when the target nucleic acid is an RNA.Reverse transcription is well known in the art and can be performed byan enzyme separate from that used for amplification (e.g., where areverse transcriptase such as MMLV reverse transcriptase is used) or bythe same enzyme (e.g., Tth polymerase or another polymerase known in theart to possess both RNA template-dependent and DNA template-dependentprimer extension abilities).

Similarly, DNA amplification is well known in the art. The methodsdescribed herein lend themselves well to standard PCR in which a pair ofselected primers flanking a target sequence directs thetemplate-dependent synthesis of copied DNA. This does not, however,exclude other methods (e.g., ligase-mediated amplification or other,isothermal, amplification methods, e.g., Self-Sustained SequenceReplication (3SR), Gingeras et al., 1990, Annales de Biologie Clinique,48(7): 498-501; Guatelli et al., 1990, Proc. Natl. Acad. Sci. U.S.A.,87:1874; see below) that can be adapted to the approach describedherein. A key element in any such alternative approach remains achievingsimilar efficiency of the amplification from a target RNA and a set ofat least two competitor nucleic acids.

3SR is an outgrowth of the transcription-based amplification system(TAS), which capitalizes on the high promoter sequence specificity andreiterative properties of bacteriophage DNA-dependent RNA polymerases todecrease the number of amplification cycles necessary to achieve highamplification levels (Kwoh et al., 1989, Proc. Natl. Acad. Sci. U.S.A.,83: 1173-1177).

In 3SR, each priming oligonucleotide contains a bacteriophage RNApolymerase binding sequence and the preferred transcriptional initiationsequence, e.g., the T7 RNA polymerase binding sequence(TAATACGACTCACTATA) and the preferred T7 polymerase transcriptionalinitiation site. The remaining sequence of each primer is complementaryto the target sequence on the molecule to be amplified.

Exemplary 3SR conditions are described herein as follows. The 3SRamplification reaction is carried out in 100 μl and contains the targetRNA, 40 mM Tris-HCl, ph 8.1, 20 mM MgCl2, 2 mM spermidine-HCl, 5 mMdithiothreitol, 80 μg/ml BSA, 1 mM dATP, 1 mM dGTP, 1 mM dTTP, 4 mMATP,4 mM CTP, 1 mM GTP, 4 mM dTTP, 4 mM ATP, 4 mM CTP, 4 mM GTP, 4 mMUTP,and a suitable amount of oligonucleotide primer (250 ng of a 57-mer;this amount is scaled up or down, proportionally, depending upon thelength of the primer sequence). Three to six attomoles of the nucleicacid target for the 3SR reactions is used. As a control for background,a 3SR reaction without any target is run in parallel. The reactionmixture is heated to 100° C. for 1 minute, and then rapidly chilled to42° C. After 1 minute, 10 units (usually in a volume of approximately 2μl) of reverse transcriptase, (e.g. avian myoblastosis virus reversetranscriptase, AMV-RT; Life Technologies/Gibco-BRL) is added. Thereaction is incubated for 10 minutes, at 42° C. and then heated to 100°C. for 1 minute. (If a 3SR reaction is performed using a single-strandedtemplate, the reaction mixture is heated instead to 65° C. for 1minute.) Reactions are then cooled to 37° C. for 2 minutes prior to theaddition of 4.6 μl of a 3SR enzyme mix, which contains 1.6 μl of AMV-RTat 18.5 units/μl, 1.0 μl T7 RNA polymerase (both e.g. from Stratagene;La Jolla, Calif.) at 100 units/μl, and 2.0 μl E. Coli RNase H at 4units/μl (e.g. from Gibco/Life Technologies; Gaithersburg, Md.). It iswell within the knowledge of one of skill in the art to adjust enzymevolumes as needed to account for variations in the specific activitiesof enzymes drawn from different production lots or supplied by differentmanufacturers. Variations can also be made to the units of the enzymesas necessary. The reaction is incubated at 37° C. for 1 hour and stoppedby freezing.

Where the progress of the amplification is to be monitored by sampling,the sampling can be performed at any stage of the 3SR reaction. Because3SR proceeds continuously at a single temperature, there are notindividual cycles at which aliquots will be withdrawn. Thus, samplingcan be performed at set times during the amplification incubationperiod, for example, every minute, every two minutes, every threeminutes, etc. Nucleic acids in the aliquots withdrawn or extruded arethen separated and nucleic acids detected, thereby permitting thegeneration of an amplification profile, from which the abundance oftarget in the initial sample can be determined.

3SR is also referred to by some as Nucleic Acid Sequence BasedAmplification, or NASBA (see for example, Compton, 1991, Nature, 350:91-92; Kievits et al., 1991, J. Virol Meth. 35: 273-286, each of whichis incorporated herein by reference).

Another method of nucleic acid amplification that is of use according tothe invention is the DNA ligase amplification reaction (LAR), which hasbeen described as permitting the exponential increase of specific shortsequences through the activities of any one of several bacterial DNAligases (Wu and Wallace, 1989, Genomics, 4: 560; Barany, 1991, Proc.Natl. Acad. Sci. USA 88: 189, each of which is incorporated herein byreference). This technique is based upon the ligation of oligonucleotideprobes. The probes are designed to exactly match two adjacent sequencesof a specific target nucleic acid. The amplification reaction isrepeated in three steps in the presence of excess probe: (1) heatdenaturation of double-stranded nucleic acid, (2) annealing of probes totarget nucleic acid, and (3) joining of the probes by thermostable DNAligase. The reaction is generally repeated for 20-30 cycles. Thesampling methods disclosed herein permit the generation of a detailedamplification profile. As with any cyclic amplification protocol, wheredesired, e.g., to establish an amplification profile, sampling can beperformed after any cycle, but preferably after each cycle.

Rolling circle amplification (RCA) is an alternative amplificationtechnology that may prove to have as large an impact as PCR. Thistechnique draws on the DNA replication mechanism of some viruses. InRCA, similar to the replication technique used by many viruses, apolymerase enzyme reads off of a single promoter around a circle ofDNA—continuously rolling out linear, concatenated copies of the circle.In such linear RCA, the reaction can run for three days, producingmillions of copies of the small circle sequence. An exponential varianthas been developed in which a second promoter displaces the doublestrands at each repeat and initiates hyperbranching in the DNAreplication, creating as many as 10¹² copies per hour.

Another amplification method that can benefit from the sampling methodsdisclosed herein is strand-displacement amplification (SDA; Walker etal., 1992, Nucleic Acids Res., 20:1691-1696; Spargo et al., 1993, Mol.Cellular Probes 7: 395-404, each of which is incorporated herein byreference). SDA uses two types of primers and two enzymes (DNApolymerase and a restriction endonuclease) to exponentially producesingle-stranded amplicons asynchronously. A variant of the basic methodin which sets of the amplification primers were anchored to distinctzones on a chip reduces primer-primer interactions. This so-called“anchored SDA” approach permits multiplex DNA or RNA amplificationwithout decreasing amplification efficiency (Westin et al., 2000, NatureBiotechnology 18: 199-204, incorporated herein by reference). SDA canbenefit from sampling and separation as described herein, as repeatedsampling permits the generation of a detailed amplification profile.

Following reverse-transcription and amplification, the methods describedherein involve the separation of nucleic acid amplification products bysize. Size separation of nucleic acids is well known, e.g., by agaroseor polyacrylamide electrophoresis or by column chromatography, includingHPLC separation. A preferred approach uses capillary electrophoresis,which is both rapid and accurate, readily achieving separation ofmolecules differing in size by only one nucleotide. Capillaryelectrophoresis uses small amounts of sample and is well-adapted fordetection by, for example, fluorescence detection. Capillaryelectrophoresis is well known in the art and is described in furtherdetail herein below.

As discussed above, amplified nucleic acids corresponding to the targetnucleic acid and competitor nucleic acids are detected after separation.The detection notes both the position of a given band of nucleic acid ofa given size and the abundance of that nucleic acid by, for example, UVabsorption or, preferably, fluorescent signal. Fluorescent nucleotidescan be incorporated into the amplified nucleic acid by simply adding oneor more such nucleotides to the amplification reaction mixture prior toor during amplification. An alternative approach is to fluorescentlylabel one or more amplification primers such that every strand amplifiedfrom that primer has at least one fluorescent label associated with it.While the methods described here are fully intended to encompass the useof fluorescently labeled nucleotide analogs for labeling the amplifiedproducts, an advantage of labeling one or more amplification primers isthat primers for different target nucleic acids can be differentiallylabeled with different fluorophores, to expand, for example, the scopeof multiplexing possible with the methods described herein. With thisapproach, additional sets of target and competitor amplicons of evensimilar size can be distinguished in the same reaction.

Following detection of amplified, separated target and competitormolecules, the methods described herein use the amounts of thecompetitors detected as a standard. Because the original concentrationsof the competitors is known, and the signal from the amplified sequenceswill be proportional to the starting amounts of each sequence, and theefficiency of amplification is similar for each of the target and thecompetitor molecules, the amount of the target nucleic acid in theoriginal sample can be determined from the amount of the competitors.The accuracy of the method is further enhanced when, as is preferred,the competitors, as internal standards, were originally present atconcentrations that flank the concentration of the target molecule.

It is noted that amplification approaches such as PCR generally exhibitkinetics such that there is a limited exponential phase of theamplification process in which the amount of amplified template isclosely proportional to the amount of original template in the reaction.The exact location of this phase in a given cycling regimen will varydepending upon factors including the target sequence, primer sequencesand the initial abundance of the target template. The methods describedherein are well adapted to determining exactly when in the cyclingregimen a given target sequence was (or is, when cycling and detectionare performed simultaneously or at least contemporaneously) beingamplified in the exponential phase. Thus, in one aspect, the methodsdescribed herein can benefit from repeated sampling during theamplification cycling regimen, coupled with separation and detection ofthe target and competitor nucleic acids in the withdrawn samples. Thedetection of, for example, fluorescently labeled target and competitoramplicons at multiple cycles during the amplification permits one togenerate a plot (most often plotted automatically) of target andcompetitor amplicon abundance versus cycle number. This approachaccurately identifies the phase for any given target or competitor atwhich the amplification is proceeding in exponential phase, which inturn permits the identification of the original quantity of the targettemplate. The addition of the internal standards represented by theknown concentrations of the longer and shorter competitors furtherenhances the accuracy of the data that can be obtained in this manner.That is, one not only has the internal standards that provide a curvefrom which to identify original concentration, but one also has thebenefit of knowing at which point in the reaction the correspondencebetween initial template and amplified product is best.

Sample withdrawal during the amplification cycling regimen can beperformed manually, or, preferably automatically, e.g., under roboticcontrol. Automated sampling can enhance the uniformity of the timing ofsample withdrawal, and can help to avoid cross-contamination that mightoccur under manual sampling conditions. Automated sampling and analysisapparatuses (including capillary electrophoresis apparatuses) aredescribed in co-pending U.S. patent application Ser. No. 10/387,286,filed Mar. 12, 2003, the entirety of which is incorporated herein byreference.

In another aspect, the quantitative approach described herein is adaptedfor multiplexing—the determination of a plurality of target nucleicacids in a given sample in a single reaction. This is preferablyachieved by selecting target amplicon and competitor amplicon sizes suchthat different sets of target and competitor amplicons, distinguishableby amplicon size, are generated for each different target nucleic acid.Alternatively, or in addition, different target amplicons can bedifferentially detected in the same reaction by using differentiallylabeled amplification primers specific for different target/competitoramplicon sets. Basic multiplex PCR approaches and the considerationsnecessary to perform them successfully are known in the art and arereadily applied to the methods described herein in which the ability toefficiently separate and detect amplicons of differing sizes fromdifferent known targets permits the detection of multiple (e.g., 2, 3,5, 10, 20, 50 or more) target signals in a single reaction. MultiplexPCR generally requires that interactions between primers specific fordifferent targets be minimized in order to reduce artifacts—that is, oneseeks to avoid the ability of any two primers being used in a reactionto hybridize to each other, instead of to their respective targetmolecules. Commonly available software packages permit the analysis andprediction of primer-primer interactions for a given set of primers.

Primer Design:

The methods described herein rely upon the use of DNA oligonucleotideprimers for the amplification of target and competitor sequences.Oligonucleotide primers for use in these methods can be designedaccording to general guidance well known in the art as described herein,as well as with specific requirements as described herein for each stepof the particular methods described.

1. General Strategies for Primer Design

Oligonucleotide primers are 5 to 100 nucleotides in length, preferablyfrom 17 to 45 nucleotides, although primers of different length are ofuse. Primers for synthesizing cDNAs are preferably 10-45 nucleotides,while primers for amplification are preferably about 17-25 nucleotides.Primers useful in the methods described herein are also designed to havea particular melting temperature (Tm) by the method of meltingtemperature estimation. Commercial programs, including Oligo™, PrimerDesign and programs available on the internet, including Primer3 andOligo Calculator can be used to calculate a Tm of a polynucleotidesequence useful according to the invention. Preferably, the Tm of anamplification primer useful according to the invention, as calculatedfor example by Oligo Calculator, is preferably between about 45 and 65°C. and more preferably between about 50 and 60° C.

Tm of a polynucleotide affects its hybridization to anotherpolynucleotide (e.g., the annealing of an oligonucleotide primer to atemplate polynucleotide). In the subject methods, it is preferred thatthe oligonucleotide primer used in various steps selectively hybridizesto a target template or polynucleotides derived from the target template(i.e., first and second strand cDNAs and amplified products). Typically,selective hybridization occurs when two polynucleotide sequences aresubstantially complementary (at least about 65% complementary over astretch of at least 14 to 25 nucleotides, preferably at least about 75%,more preferably at least about 90% complementary). See Kanehisa, M.,1984, Polynucleotides Res. 12: 203, incorporated herein by reference. Asa result, it is expected that a certain degree of mismatch at thepriming site is tolerated. Such mismatch may be small, such as a mono-,di- or tri-nucleotide. Alternatively, a region of mismatch may encompassloops, which are defined as regions in which there exists a mismatch inan uninterrupted series of four or more nucleotides. 100%complementarity is preferred for the methods described herein.

Numerous factors influence the efficiency and selectivity ofhybridization of the primer to a second polynucleotide molecule. Thesefactors, which include primer length, nucleotide sequence and/orcomposition, hybridization temperature, buffer composition and potentialfor steric hindrance in the region to which the primer is required tohybridize, are considered when designing oligonucleotide primers usefulin the methods described herein.

A positive correlation exists between primer length and both theefficiency and accuracy with which a primer will anneal to a targetsequence. In particular, longer sequences have a higher meltingtemperature (T_(M)) than do shorter ones, and are less likely to berepeated within a given target sequence, thereby minimizing promiscuoushybridization. Primer sequences with a high G-C content or that comprisepalindromic sequences tend to self-hybridize, as do their intendedtarget sites, since unimolecular, rather than bimolecular, hybridizationkinetics are generally favored in solution. However, it is alsoimportant to design a primer that contains sufficient numbers of G-Cnucleotide pairings since each G-C pair is bound by three hydrogenbonds, rather than the two that are found when A and T bases pair tobind the target sequence, and therefore forms a tighter, stronger bond.Hybridization temperature varies inversely with primer annealingefficiency, as does the concentration of organic solvents, e.g.formamide, that might be included in a priming reaction or hybridizationmixture, while increases in salt concentration facilitate binding. Understringent annealing conditions, longer hybridization probes, orsynthesis primers, hybridize more efficiently than do shorter ones,which are sufficient under more permissive conditions. Preferably,stringent hybridization is performed in a suitable buffer (for example,1X R buffer, Stratagene Catalog # 600085, 1× Pfu buffer, StratageneCatalog #200536; or 1× cloned Pfu buffer, Stratagene Catalog #200532, orother buffer suitable for other enzymes used for cDNA synthesis andamplification) under conditions that allow the polynucleotide sequenceto hybridize to the oligonucleotide primers (e.g., 95° C. for PCRamplification). Stringent hybridization conditions can vary (for examplefrom salt concentrations of less than about 1M, more usually less thanabout 500 mM and preferably less than about 200 mM) and hybridizationtemperatures can range (for example, from as low as 0° C. to greaterthan 22° C., greater than about 30° C., and (most often) in excess ofabout 37° C.) depending upon the lengths and/or the polynucleotidecomposition or the oligonucleotide primers. Longer fragments may requirehigher hybridization temperatures for specific hybridization. As severalfactors affect the stringency of hybridization, the combination ofparameters is more important than the absolute measure of a singlefactor.

The design of a primer set useful in the methods described herein can befacilitated by the use of readily available computer programs, developedto assist in the evaluation of the several parameters described aboveand the optimization of primer sequences. Examples of such programs are“PrimerSelect” of the DNAStar™ software package (DNAStar, Inc.; Madison,Wis.), OLIGO 4.0 (National Biosciences, Inc.), PRIMER, OligonucleotideSelection Program, PGEN and Amplify (described in Ausubel et al.,supra).

2. Oligonucleotide Synthesis

The oligonucleotide primers themselves are synthesized using techniquesthat are also well known in the art. Methods for preparingoligonucleotides of specific sequence include, for example, cloning andrestriction digestion of appropriate sequences and direct chemicalsynthesis. Once designed, oligonucleotides can also be prepared by asuitable chemical synthesis method, including, for example, thephosphotriester method described by Narang et al., 1979, Methods inEnzymology, 68: 90, the phosphodiester method disclosed by Brown et al.,1979, Methods in Enzymology, 68: 109, the diethylphosphoramidate methoddisclosed in Beaucage et al., 1981, Tetrahedron Letters, 22: 1859, andthe solid support method disclosed in U.S. Pat. No. 4,458,066, or byother chemical methods using either a commercial automatedoligonucleotide synthesizer (which is commercially available) or VLSIPS™technology.

Competitor RNA Design and Synthesis:

Competitor nucleic acids should be amplified by the same primer setselected for a given target RNA and have similar amplificationefficiency to the target nucleic acid with the same selected set ofprimers. The competitor nucleic acids should yield amplificationproducts, with the selected set of primers, that are distinguishable inlength from each other and from the amplification product from thetarget nucleic acid. The resolution of chosen separation techniques willnecessarily bear upon the differences in length that aredistinguishable. As noted above, differences of as little as onenucleotide are routinely achievable, although even in these instances,it may be useful to have somewhat longer lengths, in order to providebetter distinction in signal. A key consideration is having the lengthdifference long enough to be detectable by the selected method, e.g.,capillary electrophoresis, but short enough that it does notsignificantly modify the amplification efficiency relative to that ofthe target nucleic acid. That is, the amplification efficiency of thelonger or shorter competitor nucleic acid must be similar to that of thetarget nucleic acid.

As discussed above, competitor nucleic acids are characterized by thepresence of sequences which permit their amplification by the same pairof oligonucleotide primers selected to amplify a given target nucleicacid. Amplification of the competitor nucleic acid by the same pair ofprimers as used to amplify the target nucleic acid assures that theannealing efficiency of the primers to both the target and competitorsequences is the same, which is important for assuring similaramplification efficiency of the competitor and target nucleic acids.

To maintain similar amplification efficiency, it is important thatcompetitor nucleic acids (or, more accurately, their amplificationproducts) have similar T_(m) to the target nucleic acid (or itsamplification products). Methods for the estimation of T_(m) for anygiven sequence are well known in the art. T_(m) is similar if, forexample, it is within 1-2° C., but preferably within 0.5 to 1° C. oreven less difference, relative to the target nucleic acid. It ispreferred that competitor and target nucleic acids comprise at least 20nucleotides or base pairs of identical sequence. This is preferably inaddition to common primer binding sequences. The primer-bindingsequences of the target and competitor nucleic acids do not need to beidentical, but should operate to permit amplification by the sameprimers. Because differences in primer annealing efficiency affectamplification efficiency, it is most straight-forward to maintainidentity in these sequences between target and competitor sequences.

One of the most straightforward ways of generating competitor nucleicacids that will have the necessarily similar amplification efficiency tothe target nucleic acid is to modify a cloned cDNA corresponding to thetarget nucleic acid, by inserting or deleting a short (e.g., a 1-20nucleotide insertion or deletion e.g., a 5-20 nucleotide or 5-10nucleotide insertion or deletion) stretch in the target sequence itself(i.e., an internal insertion or deletion). This assures similarcharacteristics for annealing and amplification efficiency, with theonly differences being the internal insertion or deletion. Whileinsertion or deletion of a short contiguous sequence is more easilyaccomplished, the insertion or deletion encompassed by this embodimentcan also include insertion or deletion on non-contiguous nucleotides orbase pairs—that is, removal or insertion at more than one locationwithin the target sequence. For shorter target amplicon sequences, e.g.,50 to 75 nucleotides, it is beneficial to keep the difference in lengthto the shorter end of this spectrum, e.g., 1 to 5 nucleotides, as thisrepresents a smaller change in make-up of the sequence on a percentagebasis. For longer target amplicon sequences, the length difference canbe longer without having as dramatic an impact on the amplificationcharacteristics of the molecule. Even in the context of longer targetamplicon sequences, the insertion or deletion is still preferably 10nucleotides (or base pairs) or fewer, particularly where the sizeseparation will be performed with a method, e.g., CE, which is capableof resolution on the basis of as little as 1 nucleotide or base pair.

One of skill in the art will understand that one factor affectingamplification efficiency is the presence of repeat stretches of the samenucleotide, e.g., poly A, poly G, etc., which tend to reduce theefficiency of amplification relative to a similar sequence without therepeats. Thus, when considering the sequence to add, or, for thatmatter, to delete, it is best to add or delete sequence that isapproximately balanced in nucleotide composition. The sequence added ordeleted can be amino acid coding or non-coding sequence, and canoptionally comprise conventional or non-conventional nucleotides, if sodesired.

The insertion or deletion of sequence useful in generating a set ofcompetitor nucleic acids is readily achieved using site-directedmutagenesis techniques well known in the art. A number of methods areknown in the art that permit the targeted mutation of DNA sequences (seefor example, Ausubel et. al. Short Protocols in Molecular Biology (1995)3^(rd) Ed. John Wiley & Sons, Inc.). In addition, there are a number ofcommercially available kits for site-directed mutagenesis, includingboth conventional and PCR-based methods. Examples include the GeneMorphRandom mutagenesis kit (Stratagene Catalog No. 600550 or 200550),EXSITE™ PCR-Based Site-directed Mutagenesis Kit available fromStratagene (Catalog No. 200502) and the QUIKCHANGE™ Site-directedmutagenesis Kit from Stratagene (Catalog No. 200518), and the CHAMELEON®double-stranded Site-directed mutagenesis kit, also from Stratagene(Catalog No. 200509).

The measurement of amplification efficiency is described herein below.

Once competitor sequences are designed, the competitor nucleic acid foruse in the methods described herein can be generated by, for example,chemical synthesis as known in the art, PCR, or, when the competitornucleic acid is an RNA,, by in vitro transcription. The technique of invitro transcription is well known to those of skill in the art. Briefly,the sequence of interest is linked to a promoter sequence for aprokaryotic polymerase, such as the bacteriophage T7, T3 and Sp6 RNApolymerase promoter, followed by in vitro transcription of the DNAtemplate using the appropriate polymerase. The template can itself be alinear PCR product into which the promoter has been incorporated, forexample, by inclusion of the appropriate promoter sequence in one of thePCR amplification primers. Where desired, linkage to two differentpromoters, one on each end, creates the potential for also generatingthe complement of the competitor RNA.

Alternatively, a DNA sequence corresponding to a desired competitor RNAcan be inserted into a vector containing an Sp6, T3 or T7 promoter. Thevector is linearized with an appropriate restriction enzyme that digeststhe vector at a single site located downstream of the competitorsequence. Following a phenol/chloroform extraction, the DNA is ethanolprecipitated, washed in 70% ethanol, dried and resuspended in sterilewater. Regardless of the exact form of the promoter/template construct(i.e., linear PCR product or linearized vector construct), the in vitrotranscription reaction is performed by incubating the linear DNA withtranscription buffer (200 mM Tris-HCl, pH 8.0, 40 mM MgCl₂, 10 mMspermidine, 250 NaCl [T7 or T3] or 200 mM Tris-HCl, pH 7.5, 30 mM MgCl₂,10 mM spermidine [Sp6]), dithiothreitol, RNase inhibitors, each of thefour ribonucleoside triphosphates, and either Sp6, T7 or T3 RNApolymerase, e.g., for 30 min at 37° C. If it is desired to prepare alabeled polynucleotide comprising RNA, unlabeled UTP can be omitted andlabeled UTP can be included in the reaction mixture. Labels can include,for example, fluorescent or radiolabels. The DNA template is thenremoved by incubation with DNaseI. Phenol extraction can be used toremove the DNAse and polymerase, followed by precipitation andquantitation of the RNA, e.g., by UV absorption and/or byelectrophoresis and visualization relative to known standards.

Polymerase Chain Reaction:

PCR provides a well-established method for rapidly amplifying aparticular DNA sequence by using multiple cycles of DNA replicationcatalyzed by a thermostable, DNA-dependent DNA polymerase to amplify thetarget sequence of interest. PCR requires the presence of a targetnucleic acid sequence to be amplified, two single strandedoligonucleotide primers flanking the sequence to be amplified, a DNApolymerase, deoxyribonucleoside triphosphates, a buffer and salts.

PCR is described in Mullis and Faloona, 1987, Methods Enzymol., 155:335, incorporated herein by reference, as well as in U.S. Pat. Nos.4,683,202, 4,683,195 and 4,800,159, each of which is also incorporatedherein by reference. Reaction conditions for the amplification of achosen target sequence can be readily selected or determined with aminimum of experimentation by one of ordinary skill in the art. Numerousvariations on the basic theme are also known to those of skill in theart.

The length and temperature of each step of a PCR cycle (denaturation,primer annealing, and extension), as well as the number of cycles, areadjusted according to the stringency requirements in effect. Annealingtemperature and timing are determined both by the efficiency with whicha primer is expected to anneal to a template and the degree of mismatchthat is to be tolerated. The ability to optimize the stringency ofprimer annealing conditions is well within the knowledge of one ofordinary skill in the art. An annealing temperature of between 30° C.and 72° C. is most often used. Initial denaturation of the templatemolecules normally occurs at between 92° C. and 99° C., e.g., for 4minutes, followed by 10-40 cycles consisting of denaturation (94-99° C.for 15 seconds to 1 minute), annealing (temperature determined asdiscussed above; 30 seconds to 2 minutes), and extension (72° C. for 30seconds to 1 minute; this is optimal for Taq polymerase—one of skill inthe art will know or can easily determine suitable extension conditionsfor different thermostable polymerases). Depending upon the intended useof the product, a final extension step is often carried out for a longertime, e.g., 4 minutes at 72° C., and may be followed by an indefinite(0-24 hour) storage at 4° C.

Polymerases:

A wide variety of DNA polymerases can be used in the methods describedherein. Suitable DNA polymerases for use in the subject methods may ormay not be thermostable, although thermostable polymerases are obviouslypreferred for the embodiments using thermocycling for amplification.Known conventional DNA polymerases include, for example, Pyrococcusfuriosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1,provided by Stratagene), Pyrococcus woesei (Pwo) DNA polymerase(Hinnisdaels et al., 1996, Biotechniques, 20:186-8, provided byBoehringer Mannheim), Thermus thermophilus (Tth) DNA polymerase (Myersand Gelfand 1991, Biochemistry 30:7661), Bacillus stearothermophilus DNApolymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475 : 32),Thermococcus litoralis (Tli) DNA polymerase (also referred to as VentDNA polymerase, Cariello et al., 1991, Polynucleotides Res, 19: 4193,provided by New England Biolabs), Vent exó (New England Biolabs), 9° NmDNA polymerase (discontinued product from New England Biolabs),Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998, Braz J.Med. Res, 31: 1239), Thermus aquaticus (Taq) DNA polymerase (Chien etal., 1976, J. Bacteoriol, 127: 1550), Pyrococcus kodakaraensis KOD DNApolymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63: 4504),JDF-3 DNA polymerase (from thermococcus sp. JDF-3, Patent application WO0132887), Pyrococcus GB-D (PGB-D) DNA polymerase (also referred asDeep-Vent DNA polymerase, Juncosa-Ginesta et al., 1994, Biotechniques,16: 820, provided by New England Biolabs), UITma DNA polymerase (fromthermophile Thermotoga maritima; Diaz and Sabino, 1998, Braz J. Med.Res. 31: 1239; provided by PE Applied Biosystems), Tgo DNA polymerase(from thermococcus gorgonarius, provided by Roche MolecularBiochemicals), E. coli DNA polymerase I (Lecomte and Doubleday, 1983,Polynucleotides Res. 11: 7505), T7 DNA polymerase (Nordstrom et al.,1981, J. Biol. Chem. 256: 3112), and archaeal DP1/DP2 DNA polymerase II(Cann et al., 1998, Proc. Natl. Acad. Sci. USA 95:14250-5).

For thermocyclic reactions, the polymerases are preferably thermostablepolymerases such as Taq, Deep Vent, Tth, Pfu, Vent, and UITma, each ofwhich are readily available from commercial sources. Similarly, guidancefor the use of each of these enzymes can be readily found in any of anumber of protocols found in guides, product literature, the Internet(see, for example, www.alkami.com), and other sources.

For non-thermocyclic reactions, and in certain thermocyclic reactions,the polymerase will often be one of many polymerases commonly used inthe field, and commercially available, such as DNA pol 1, Klenowfragment, T7 DNA polymerase, and T4 DNA polymerase. In applicationsinvolving transcription, a number of RNA polymerases are alsocommercially available, such as T7 RNA polymerase and SP6 RNApolymerase. Guidance for the use of such polymerases can readily befound in product literature and in general molecular biology guides suchas Sambrook or Ausubel, both supra.

Polymerases can incorporate labeled (e.g., fluorescent) nucleotides ortheir analogs during synthesis of polynucleotides. See, e.g., Hawkins etal., U.S. Pat. No. 5,525,711, where the use of nucleotide analogs whichare incorporatable by Taq is described.

As described above, the amplification reactions required for the methodsdescribed herein can generally be carried out using standard reactionconditions and reagents unless otherwise specified. Such reagents andconditions are well known to those of skill in the art, and aredescribed in numerous references and protocols. See, e.g. Innis supra;Sambrook, supra.; Ausubel, et al., eds. (1996) Current Protocols inMolecular Biology Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc. Also, see,Mullis et al., (1987) U.S. Pat. No. 4,683,202, and Arnheim & Levinson(1990) C&EN 6-47, The Journal Of NIH Research (1991) 3: 81-94; Kwoh etal. (1989) Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990)Proc. Natl. Acad. Sci. USA 87: 1874; Lomell et al. (1989) J. Clin. Chem35: 1826; Landegren et al., (1988) Science 241: 1077-1080; Van Brunt(1990) Biotechnology 8: 291-294; Wu and Wallace, (1989) Gene 4: 560;Barringer et al. (1990) Gene 89: 117, and Sooknanan and Malek (1995)Biotechnology 13: 563-564.

Amplification Efficiency:

As discussed above, the amplification efficiency of the competitornucleic acid should be similar to that of the target nucleic acid. Inone aspect, amplification efficiency is expressed as the foldamplification per PCR cycle, represented as a fraction or percentagerelative to perfect doubling. A 100% or 1.0 amplification efficiencywould refer to perfect doubling.

One way to monitor amplification efficiency is to measure the thresholdcycle number (Ct) at which signal intensity of PCR product reaches a setthreshold value (for example 10 standard deviations of background valueof signal intensity) for an amplified product. Samples are withdrawn at,for example, each cycle during the amplification regimen and analyzedfor the amount of target amplicon. Comparison of Ct for equal startingamounts of two different amplification templates, e.g., a target RNA anda competitor RNA will determine whether the amplification efficiency issimilar. To enhance accuracy, the determination can be performed atseveral different equal starting concentrations of target and competitorRNAs. Amplification efficiency is considered “similar” if the thresholdcycle, Ct, is the same for equal starting amounts of eachcompetitor/target set.

Ct is linked to the initial copy number or concentration of starting DNAby a simple mathematical equation:Log(copy number)=aC _(t) +b, where a and b are constants.

Therefore, by measuring C_(t) for the fragments of the same geneoriginating from two different samples, the original concentration ofthis gene in these samples can be easily evaluated. Alternatively,amplification efficiency is monitored by measuring the amount ofamplification product (e.g., by fluorescence intensity or labelincorporation) at successive cycles, calculating efficiency using theformula E=(P_(n+1)−P_(n))/(P_(n)−P_(n−1)), where P is the amount ofamplification product at cycle n.

While the similarity in amplification efficiencies will ultimately bedetermined empirically, the maintenance of target sequence identity inthe competitors, except for an insertion or deletion necessary togenerate a detectable difference in length relative to the target, willassist in achieving similar efficiencies.

It is known that the presence of various contaminants in a nucleic acidsample preparation can have an effect on amplification efficiency. Anadvantage of the methods described herein is that any such contaminantwill most likely affect the efficiency of amplification of both thecompetitor and target amplicons to a similar degree, because each ofthese amplicons is generated in the same reaction. This will tend toreduce the impact of any such inhibition of efficient amplification.

Preparation of Samples

A target polynucleotide of the present invention may be single- ordouble-stranded, and it may be DNA (e.g., gDNA or cDNA), RNA, apolynucleotide comprising both deoxyribo- and ribonucleotides, or apolynucleotide comprising deoxyribonucleotides, ribonucleotides, and/oranalogs and derivatives thereof. Where one wishes to determine the levelof expression of a gene, the target polynucleotide is an RNA molecule,e.g., an mRNA molecule.

Before the amplification reaction, the target polynucleotide may beobtained in suitable quantity and quality for the chosen amplificationmethod to be used. For example, in some instances, the samples containsuch a low level of target polynucleotide that it is useful to conduct apre-amplification reaction to increase the concentration of the targetpolynucleotide. If samples are to be amplified, amplification istypically conducted using the polymerase chain reaction (PCR) accordingto known procedures. In some embodiments, it may be preferred to addknown quantities of the competitor nucleic acids to a biological sampleprior to co-isolation of competitor and test nucleic acids in thesample.

Guidance for the preparation of a sample containing a targetpolynucleotide can be found in a multitude of sources, including PCRProtocols, A Guide to Methods and Applications (Innis et al., supra;Sambrook et al., supra; Ausubel et al., supra). Any such method can beused in methods described herein. Typically, these methods involve celllysis, followed by purification of polynucleotides by methods such asphenol/chloroform extraction, electrophoresis, and/or chromatography.Often, such methods include a step wherein the polynucleotides areprecipitated, e.g. with ethanol, and resuspended in an appropriatebuffer for addition to a PCR or similar reaction.

In certain embodiments, two or more target polynucleotides from one ormore sample sources are analyzed in a single reaction. In someapplications, a single polynucleotide from a multitude of sources may besynthesized to screen for the presence or absence of a particularsequence difference. In other applications, a plurality ofpolynucleotides may be amplified from a single sample or individual,thereby allowing the assessment of a variety of polynucleotides in asingle individual, e.g., to simultaneously screen for a multitude ofdisease markers in an individual. Any of the above applications can beeasily accomplished using the methods described herein.

A reaction mixture may comprise one target polynucleotide, or it maycomprise two or more target polynucleotides. The present method allowsfor simultaneous analysis of two or more polynucleotides obtained from aplurality of samples, i.e., multiplex analysis.

In one aspect of the invention, a nucleic acid sample may be derivedfrom a sample from an animal suffering from an infectious disease (e.g.,a disease of bacterial, fungal, viral or parasitic origin) and anothersample of may be from an animal not suffering from an infectiousdisease. In another aspect, a nucleic acid sample may be may be derivedfrom an animal suffering from cancer and another may be derived from ananimal not suffering from cancer. In another aspect, one an a nucleicacid sample may be may be obtained from a cancerous animal tissue andanother may be obtained from a noncancerous animal tissue, which tissuesmay both be obtained from the same animal. In another aspect, a nucleicacid sample may be may be from an animal suffering from a geneticdisease and another sample may be from an animal not suffering from agenetic disease. In another aspect, a nucleic acid sample may be may beobtained from a pathogenic microorganism and another library or samplemay be obtained from a non-pathogenic microorganism. In another aspect,a nucleic acid sample may be derived from an organism expressing anenzyme, and another may be derived from an organism not expressing anenzyme. Other suitable sources of a nucleic acid sample will be apparentto one of ordinary skill in the art.

Once the starting cells, tissues, organs or other samples are obtained,nucleic acids (including RNA and/or DNA) can be prepared therefrom bymethods that are well-known in the art.

RNA can be purified, for example, from tissues according to thefollowing method. Following removal of the tissue of interest, pieces oftissue of ≦2 g are cut and quick frozen in liquid nitrogen, to preventdegradation of RNA. Upon the addition of a suitable volume ofguanidinium solution (for example 20 ml guanidinium solution per 2 g oftissue), tissue samples are ground in a tissuemizer with two or three10-second bursts. To prepare tissue guanidinium solution (1 L) 590.8 gguanidinium isothiocyanate is dissolved in approximately 400 mlDEPC-treated H₂O. 25 ml of 2 M Tris-HCl, pH 7.5 (0.05 M final) and 20 mlNa₂EDTA (0.01 M final) is added, the solution is stirred overnight, thevolume is adjusted to 950 ml, and 50 ml 2-ME is added.

Homogenized tissue samples are subjected to centrifugation for 10 min at12,000×g at 120 C. The resulting supernatant is incubated for 2 min at650 C in the presence of 0.1 volume of 20% Sarkosyl, layered over 9 mlof a 5.7M CsCl solution (0.1 g CsCl/ml), and separated by centrifugationovernight at 113,000×g at 220 C. After careful removal of thesupernatant, the tube is inverted and drained. The bottom of the tube(containing the RNA pellet) is placed in a 50 ml plastic tube andincubated overnight (or longer) at 40 C in the presence of 3 ml tissueresuspension buffer (5 mM EDTA, 0.5% (v/v) Sarkosyl, 5% (v/v) 2-ME) toallow complete resuspension of the RNA pellet. The resulting RNAsolution is extracted sequentially with 25:24:1phenol/chloroform/isoamyl alcohol, followed by 24:1 chloroform/isoamylalcohol, precipitated by the addition of 3 M sodium acetate, pH 5.2, and2.5 volumes of 100% ethanol, and resuspended in DEPC water (Chirgwin etal., 1979, Biochemistry, 18: 5294).

Alternatively, RNA can be isolated from tissues according to thefollowing single step protocol. The tissue of interest is prepared byhomogenization in a glass teflon homogenizer in 1 ml denaturing solution(4M guanidinium thiosulfate, 25 mM sodium citrate, pH 7.0, 0.1M 2-ME,0.5% (w/v) N-laurylsarkosine) per 100 mg tissue. Following transfer ofthe homogenate to a 5-ml polypropylene tube, 0.1 ml of 2 M sodiumacetate, pH 4, 1 ml water-saturated phenol, and 0.2 ml of 49:1chloroform/isoamyl alcohol are added sequentially. The sample is mixedafter the addition of each component, and incubated for 15 min at 0-4□Cafter all components have been added. The sample is separated bycentrifugation for 20 min at 10,000×g, 4□C, precipitated by the additionof 1 ml of 100% isopropanol, incubated for 30 minutes at −20□C andpelleted by centrifugation for 10 minutes at 10,000×g, 4□C. Theresulting RNA pellet is dissolved in 0.3 ml denaturing solution,transferred to a microfuge tube, precipitated by the addition of 0.3 mlof 100% isopropanol for 30 minutes at −20□C, and centrifuged for 10minutes at 10,000×g at 4□C. The RNA pellet is washed in 70% ethanol,dried, and resuspended in 100-200 μl DEPC-treated water or DEPC-treated0.5% SDS (Chomczynski and Sacchi, 1987, Anal. Biochem., 162: 156).

Kits and reagents for isolating total RNAs are commercially availablefrom various companies, for example, RNA isolation kit (Stratagene, LaLola, Calif., Cat # 200345); PicoPure™ RNA Isolation Kit (Arcturus,Mountain View, Calif., Cat # KIT0202); RNeasy Protect Mini, Midi, andMaxi Kits (Qiagen, Cat # 74124).

In some embodiments, total RNAs are used in the subject method forsubsequent analysis, e.g., for reverse transcription. In otherembodiments, mRNAs are isolated from the total RNAs or directly from thesamples to use for reverse transcription. Kits and reagents forisolating mRNAs are commercially available from, e.g., Oligotex mRNAKits (Qiagen, Cat # 70022).

Labeled Nucleotides

The methods described herein can benefit from the use of labelsincluding, e.g., fluorescent labels. In one aspect, the fluorescentlabel can be a label or dye that intercalates into or otherwiseassociates with amplified (usually double-stranded) nucleic acidmolecules to give a signal. One stain useful in such embodiments is SYBRGreen (e.g., SYBR Green I or II, commercially available from MolecularProbes Inc., Eugene, Oreg.). Others known to those of skill in the artcan also be employed in the methods described herein. An advantage ofthis approach is reduced cost relative to the use of, for example,labeled nucleotides. Nonetheless, it may also be preferred that thelabel will be incorporated by attachment to a labeled nucleotide ornucleotide analog that is a substrate for the polymerizing enzyme. Labelcan alternatively be attached to an amplification primer. As taughtabove, a labeled nucleotide can be a fluorescent dye-linked nucleotide,or it can be an intrinsically fluorescent nucleotide. In one embodimentof the methods described herein, a conventional deoxynucleotide linkedto a fluorescent dye is used. Non-limiting examples of some usefullabeled nucleotide are listed in Table 1. TABLE 1 Examples of labelednucleotides Fluorescein Labeled Fluorophore Labeled Fluorescein - 12 -dCTP Eosin - 6 - dCTP Fluorescein - 12 - dUTP Coumarin - 5 -ddUTPFluorescein - 12 - dATP Tetramethylrhodamine - 6 - dUTP Fluorescein -12 - dGTP Texas Red - 5 - dATP Fluorescein - N6 - dATP LISSAMINE ™ -rhodamine - 5 - dGTP FAM Labeled TAMRA Labeled FAM - dUTP TAMRA - dUTPFAM - dCTP TAMRA - dCTP FAM - dATP TAMRA - dATP FAM - dGTP TAMRA - dGTPROX Labeled JOE Labeled ROX - dUTP JOE - dUTP ROX - dCTP JOE - dCTPROX - dATP JOE - dATP ROX - dGTP JOE - dGTP R6G Labeled R110 LabeledR6G - dUTP R110 - dUTP R6G - dCTP R110 - dCTP R6G - dATP R110 - dATPR6G - dGTP R110 - dGTP BIOTIN Labeled DNP Labeled Biotin - N6 - dATPDNP - N6 - dATP

Fluorescent dye-labeled nucleotide can be purchased from commercialsources. Labeled polynucleotides nucleotide can also be prepared by anyof a number of approaches known in the art.

Fluorescent dyes useful as detectable labels are well known to thoseskilled in the art and numerous examples can be found in the Handbook ofFluorescent Probes and Research Chemicals 6th Edition, Richard Haugland,Molecular Probes, Inc., 1996 (ISBN 0-9652240-0-7).

Preferably, fluorescent dyes are selected for compatibility withdetection on an automated capillary electrophoresis apparatus and thusshould be spectrally resolvable and not significantly interfere withelectrophoretic analysis. Examples of suitable fluorescent dyes for useas detectable labels can be found in among other places, U.S. Pat. Nos.5,750,409; 5,366,860; 5,231,191; 5,840,999; 5,847,162; 4,439,356;4,481,136; 5,188,934; 5,654,442; 5,840,999; 5,750,409; 5,066,580;5,750,409; 5,366,860; 5,231,191; 5,840,999; 5,847,162; 5,486,616;5,569,587; 5,569,766; 5,627,027; 5,321,130; 5,410,030; 5,436,134;5,534,416; 5,582,977; 5,658,751; 5,656,449; 5,863,753; PCT PublicationsWO 97/36960; 99/27020; 99/16832; European Patent EP 0 050 684; Sauer etal, 1995, J. Fluorescence 5: 247-261; Lee et al., 1992, Nucl. Acids Res.20: 2471-2483; and Tu et al., 1998, Nucl. Acids Res. 26: 2797-2802, allof which are incorporated herein in their entireties.

Nucleotide can be modified to include functional groups, such as primaryand secondary amines, hydroxyl, nitro and carbonyl groups, forfluorescent dye linkage (see Table 2). TABLE 2 Functional Group ReactionProduct Amine dye - isothiocyanates Thiourea Amine dye - succinimidylester Carboxamide Amine dye - sulfonyl chloride Sulphonamide Amine dye -aldehyde Alkylamine Ketone dye - hydrazides Hydrazones Ketone dye -semicarbazides Hydrazones Ketone dye - carbohydrazides Hydrazones Ketonedye - amines Alkylamine Aldehyde dye - hydrazides Hydrazones Aldehydedye - semicarbazides Hydrazones Aldehyde dye - carbohydrazidesHydrazones Aldehyde dye - amines Alkylamine Dehydrobutyrine dye -sulphydryl Methyl lanthionine Dehydroalanine dye - sulphydrylLanthionine

Useful fluorophores include, but are not limited to: Texas Red™ (TR),Lissamine™ rhodamine B, Oreg. Green™ 488 (2′,7′-difluorofluorescein),carboxyrhodol and carboxyrhodamine, Oreg. Green™ 500, 6-JOE(6-carboxy-4′,5′-dichloro-2′,7′-5 dimethyoxyfluorescein, eosin F3S(6-carobxymethylthio-2′,4′, 5′,7′-tetrabromo-trifluorofluorescein),cascade blue™ (CB), aminomethylcoumarin (AMC), pyrenes, dansyl chloride(5-dimethylaminonaphthalene-1-sulfonyl chloride) and othernapththalenes, PyMPO, ITC(1-(3-isothiocyanatophenyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl)pyridiniumbromide), coumarin, fluorescein, tetrachlorofluorescein,hexachlorofluorescein, Lucifer yellow, rhodamine, BODIPY,tetramethylrhodamine, Cy3, Cy5, Cy7, eosine, and ROX. Combinationfluorophores such as fluorescein-rhodamine dimers, described, forexample, by Lee et al. (1997), Polynucleotides Research 25:2816, arealso suitable. Fluorophores may be chosen to absorb and emit in thevisible spectrum or outside the visible spectrum, such as in theultraviolet or infrared ranges. Suitable fluorescent dye labels arecommercially available from Molecular Probes, Inc., Eugene, Oreg., USand Research Organics, Inc., Cleveland, Ohio, US, among other sources,and can be found in the Handbook of Fluorescent Probes and ResearchChemicals 6th Edition, Richard Haugland, Molecular Probes, Inc., 1996(ISBN 0-9652240-0-7).

A labeled nucleotide useful in the methods described herein includes anintrinsically fluorescent nucleotide known in the art, e.g., the novelfluorescent nucleoside analogs as described in U.S. Pat. No. 6,268,132B1(the entirety is hereby incorporated by reference). The fluorescentanalogs of the U.S. Pat. No. 6,268,132B1 are of three general types: (A)C-nucleoside analogs; (B) N-nucleoside analogs; and (C)N-azanucleotideand N-deazanucleotide analogs. All of these compounds have threefeatures in common: 1) they are structural analogs of the commonnucleosides capable of replacing naturally occurring nucleosides inenzymatic or chemical synthesis of oligonucleotides; 2) they arenaturally fluorescent when excited by light of the appropriatewavelength(s) and do not require additional chemical or enzymaticprocesses for their detection; and 3) they are spectrally distinct fromthe nucleosides commonly encountered in naturally occurring DNA. Atleast 125 specific compounds have been identified in U.S. Pat. No.6,268,132B1. These compounds, which have been characterized according totheir class, structure, chemical name, absorbance spectra, emissionspectra, and method of synthesis, are tabulated as shown in FIGS.21A-21F-1 of the U.S. Pat. No. 6,268,132B1.

The labeled nucleotide as described herein also includes, but is notlimited to, fluorescent N-nucleosides and fluorescent structuralanalogs. Formycin A (generally referred to as Formycin), theprototypical fluorescent nucleoside analog, was originally isolated asan antitumor antibiotic from the culture filtrates of Nocardiainterforma (Hori et al. [1966] J. Antibiotics, Ser. A 17:96-99) and itsstructure identified as 7-amino-3-b-D-ribafuranosyl (1H-pyrazolo-[4,3d]pyrimidine)) (FIGS. 5 and 6). This antibiotic, which has also beenisolated from culture broths of Streptomyces lavendulae (Aizawa et al.[1965] Agr. Biol. Chem. 29:375-376), and Streptomyces gummaensis(Japanese Patent No. 10,928, issued in 1967 to Nippon Kayaku Co., Ltd.),is one of numerous microbial C-ribonucleoside analogs of theN-nucleosides commonly found in RNA from all sources. The othernaturally-occurring C-ribonucleosides which have been isolated frommicroorganisms (FIG. 4) include formycin B (Koyama et al.

Tetrahedron Lett. 597-602; Aizawa et al., supra; Umezawa et al. [1965]Antibiotics Ser. A 18:178-181), oxoformycin B (Ishizuka et al. [1968] J.Antibiotics 21:1-4; Sawa et al. [1968] Antibiotics 21:334-339),pseudouridine (Uematsu and Suahdolnik [1972] Biochemistry 11:4669-4674),showdomycin (Darnall et al. [1967] PNAS 57:548-553), pyrazomycin (Sweenyet al. [1973] Cancer Res. 33:2619-2623), and minimycin (Kusakabe et al.[1972] J. Antibiotics 25:44-47). Formycin, formycin B, and oxoformycin Bare pyrazolopyrimidinenucleosides and are structural analogs ofadenosine, inosine, and hypoxanthine, respectively; a pyrazopyrimidinestructural analog of guanosine obtained from natural sources has notbeen reported in the literature. A thorough review of the biosynthesisof these compounds is available in Ochi et al. (1974) J. Antibioticsxxiv:909-916. The entirety of each reference is here by incorporated byreference.

Separation and Detection of Amplified Products:

Methods for detecting the presence or amount of polynucleotides are wellknown in the art and any of them can be used in the methods describedherein so long as they are capable of separating individualpolynucleotides by at least the difference in length between competitorand target amplicons. The separation technique used should permitresolution of sequences from 25 to 1000 nucleotides or base pair, longand have a resolution of 10 nucleotides or base pairs or better. Theseparation can be performed under denaturing or under non-denaturing ornative conditions—i.e., separation can be performed on single- ordouble-stranded Nas. It is preferred that the separation and detectionpermits detection of length differences as small as one nucleotide. Itis further preferred that the separation and detection can be done in ahigh-throughput format that permits real time or contemporaneousdetermination of amplicon abundance in a plurality of reaction aliquotstaken during the cycling reaction. Useful methods for the separation andanalysis of the amplified products include, but are not limited to,electrophoresis (e.g., capillary electrophoresis (CE)), chromatography(dHPLC), and mass spectrometry.

In one embodiment, CE is a preferred separation means because itprovides exceptional separation of the polynucleotides in the range ofat least 10-1,000 base pairs with a resolution of a single base pair. CEcan be performed by methods well known in the art, for example, asdisclosed in U.S. Pat. Nos. 6,217,731; 6,001,230; and 5,963,456, whichare incorporated herein by reference. High-throughput CE apparatuses areavailable commercially, for example, the HTS9610 High throughputanalysis system and SCE 9610 fully automated 96-capillaryelectrophoresis genetic analysis system from Spectrumedix Corporation(State College, Pa.); P/ACE 5000 series and CEQ series from BeckmanInstruments Inc (Fullerton, Calif.); and ABI PRISM 3100 genetic analyzer(Applied Biosystems, Foster City, Calif.). Near the end of the CEcolumn, in these devices the amplified DNA fragments pass a fluorescentdetector which measures signals of fluorescent labels. These apparatusesprovide automated high throughput for the detection offluorescence-labeled PCR products.

The employment of CE in the methods described herein permits higherproductivity compared to conventional slab gel electrophoresis. Theseparation speed is limited in slab gel electrophoresis because of theheat produced when the high electric field is applied to the gel. Sinceheat elimination is very rapid from the large surface area of acapillary, a higher electric field can be applied in capillaryelectrophoresis, thus accelerating the separation process. By using acapillary gel, the separation speed is increased about 10 fold overconventional slab-gel systems.

With CE, one can also analyze multiple samples at the same time, whichis essential for high-throughput. This is achieved, for example, byemploying multi-capillary systems. In some instances, the detection offluorescence from DNA bases may be complicated by the scattering oflight from the porous matrix and capillary walls. However, a confocalfluorescence scanner can be used to avoid problems due to lightscattering (Quesada et al., 1991, Biotechniques 10: 616-25).

In one embodiment, the methods described herein measure the amount(i.e., copy number) of a particular target nucleic acid (e.g., DNA orRNA) contained in the sample used as template for amplification.

In another embodiment, differences in gene expression, rather than theexact copy numbers of the target polynucleotide contained in the sampleis measured. The detected signal strength following size separation canbe recorded for each of the at least two competitors and the target RNAin two separate samples and used to determine the relative ratio of thetarget polynucleotide from two samples. A threshold cycle number (Ct) iscalculated as a cycle number at which signal intensity of PCR productwill reach a set threshold value (for example 10 standard deviations ofbackground value of signal intensity) for an amplified product.Operational differential expression of a particular target is determinedas a difference in threshold cycle number (Ct) for this target in two(or more) samples, of more than one cycle in value. In addition to thequantitation achieved by reference to the signals from the at least twocompetitor RNAs in such an embodiment, the threshold cycle number for agiven target in a given reaction can be further used to derive copynumber for the target polynucleotide and to measure the difference inthe expression by a ratio of copy numbers for the target in two or moresamples.

EXAMPLES

Various embodiments of the invention are exemplified in the followingnon-limiting examples.

1) Quantitative measurement of a target RNA.

To determine quantity of target RNA “X”, to the sample of RNA containingspecific RNA “X” two quantitative competitive standards A and B areadded in the quantities of 20 copies of standard A and 2000 copies ofstandard B.

Quantitative competitive standards A and B are designed in to beco-amplified with target sequence X using the same oligonucleotideprimers, and to possess similar (preferably the same) amplificationefficiency as target RNA X, and to produce amplified products thatdiffer in size from amplified target RNA X and from each other. Forexample, target sequence will be amplified in RT-PCR reaction to producea DNA fragment of 150 bases, standard A will be amplified to produce afragment of 140 bases, and standard B will be amplified to produce afragment of 130 bases; sequences of amplified products will beessentially identical with exception of absence of 10 bases in sequenceA and the presence of an additional 10 bases in sequence B when comparedto the sequence X.

Amplification of target gene and quantitative standards is conducted byRT-PCR as follows:

a) Reverse transcriptase and RT primer are added to RNA to performreverse transcription (RT) under standard conditions. The RT primer ispreferably designed to prime DNA synthesis upstream of the sequencewhich will be used in PCR amplification. In this approach, following RT,DNA polymerase and a pair of PCR primers are added to conduct PCRamplification under standard conditions.

b) Alternatively, reverse transcriptase, PCR primers and DNA polymeraseare added to conduct one-step RT-PCR amplification.

Following RT and PCR, separation of amplified PCR products is performedby CE.

In one of the simplest methods, the signal measured for DNA peaks at 140and 130 bases (corresponding to standards A and B) will be used tocreate a calibration corresponding to the starting amounts of 20 and2000 copies of the standards. The signal generated by unknown amount oftarget sequence X will be measured at the peak of 150 bases andnormalized to the measured signals and starting amounts of standards Aand B. Results are shown schematically, for example, in FIG. 1.

2) Quantitative measurement of a target RNA.

To extend the calibrated concentration range (to cover possible highconcentrations of target X) additional standard C (producing amplifiedproduct of 160 bases) can be added at 200000 copies.

The amplification and separation procedure will be conducted as inExample 1 with one modification. Since the dynamic range of detection ofmost current CE systems is in the range of 100-1000X, it may not bepossible to observe signals from low copies (e.g. 20) and high copies(e.g. 200000) on the same electrophoregram. To overcome this limitation,the amplification reaction will be sampled for CE separation at multiplecycles:

For example: at cycle 15, where signal from target could be normalizedto the amplified high copy standard C; at cycle 22 where signal fromtarget could be normalized to the amplified medium copy standard B; atcycle 30 where signal from target could be normalized to the amplifiedlow copy standard A.

3) Multiplex measurement of target RNAs.

Multiplex detection of at least two genes X and Y is performed where thereaction is supplemented with standards A and B for target X andstandards E and F for target Y, and two separate primer pairs foramplification of targets X and Y correspondingly. Amplification productsgenerated from standards E and F and target Y differ in size fromamplification products generated from each of target X and standards Aand B.

The assay can be conducted using the procedure from Example 1 or 2, withCE size separation providing discrimination between the amplificationproducts of.

The present invention is not to be limited in scope by the exemplifiedembodiments which are intended as illustrations of single aspects of theinvention. Various modifications of the invention in addition to thosedescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying figures. Such modificationsare intended to fall within the scope of the appended claims. Allpublications cited herein are incorporated by reference in theirentirety.

1. A method of determining the level of a target nucleic acid in anucleic acid sample, the method comprising: a) for a given targetnucleic acid, selecting a pair of first and second amplification primersthat will generate a target amplicon of a first known length when saidtarget RNA is subjected to amplification using said pair of primers; b)providing at least two nucleic acid competitor molecules: i) a firstcompetitor nucleic acid molecule comprising said target nucleic acidsequence plus or minus an internal insertion or deletion of knownlength, wherein said first nucleic acid competitor is a substrate foramplification by said pair of first and second amplification primers,such amplification generating an amplicon of a second known length thatis longer or shorter than said target amplicon by the length of saidinternal insertion or deletion; ii) a second nucleic acid competitormolecule comprising said target nucleic acid sequence plus or minus aninternal insertion or deletion of known length, wherein said secondnucleic acid competitor is a substrate for amplification by said pair offirst and second amplification primers, such amplification generating anamplicon of a third known length, different from said second knownlength, that is longer or shorter than said target amplicon by thelength of said internal insertion or deletion; wherein the amplificationof said target nucleic acid and said first and second nucleic acidcompetitor molecules proceeds with similar efficiency; c) combining atest nucleic acid sample with said first and second nucleic acidcompetitor molecules, wherein the respective competitors are added atfirst and second known concentrations, said concentrations beingdifferent by at least one order of magnitude; d) performingreverse-transcription and target sequence amplification on the combinednucleic acid sample of step (c) using said pair of first and secondamplification primers; e) separating the products of amplification step(d); and f) detecting the amounts of the target amplicon of said firstknown length, the competitor amplicon of said second known length andthe competitor amplicon of said third known length, wherein when theamount of one said competitor amplicon is greater than the amount ofsaid target amplicon and the amount of the other said target amplicon isless than the amount of said target amplicon, the concentration of saidtarget nucleic acid in said sample is determined.
 2. The method of claim1 wherein the amplicons generated by said first and second competitornucleic acids are shorter than the amplicon generated from said targetnucleic acid, each by the length of an internal deletion.
 3. The methodof claim 1 wherein the amplicons generated by said first and secondcompetitor nucleic acids are longer than the amplicon generated fromsaid target nucleic acid, each by the length of an internal insertion.4. The method of claim 1 wherein the amplicon generated by said firstcompetitor nucleic acid is longer than the amplicon generated by saidtarget nucleic acid by the length of an internal insertion, and whereinthe amplicon generated by said second competitor nucleic acid is shorterthan the amplicon generated by said target nucleic acid by the length ofan internal deletion.
 5. The method of claim 1 wherein said targetnucleic acid is a DNA.
 6. The method of claim 5 wherein said competitornucleic acids are DNAs.
 7. The method of claim 1 wherein said targetnucleic acid is an RNA, and wherein said method comprises before step(d), of reverse-transcribing said target nucleic acid.
 8. The method ofclaim 7 wherein said competitor nucleic acids are RNAs.
 9. The method ofclaim 7 wherein said method measures gene expression of said target RNA.10. The method of claim 1 wherein said competitor nucleic acids areadded to a test sample prior to preparation of nucleic acid from saidtest ample.
 11. The method of claim 1 wherein said separating of step(e) is performed by capillary electrophoresis.
 12. The method of claim 1wherein said step of detecting comprises detection of fluorescent labelincorporated into said amplicons during amplification.
 13. The method ofclaim 1 wherein said step of detecting comprises detection of afluorescent dye that binds double-stranded amplification products. 14.The method of claim 13 wherein said fluorescent dye comprises a SYBRGREEN dye.
 15. The method of claim 1 wherein said concentrations of saidfirst and second competitor nucleic acids differ by at least two ordersof magnitude.
 16. The method of claim 1 wherein said concentrations ofsaid first and second competitor nucleic acids differ by at least threeorders of magnitude.
 17. The method of claim 1 wherein saidamplification is performed in the presence of a fluorescently-labelednucleotide, such that amplification products are fluorescently labeled.18. The method of claim 1 wherein a plurality of aliquots of theamplification reaction of step (d) are taken during the amplificationregimen, wherein said aliquots, upon separation and detection of nucleicacid permit the generation of an amplification profile for each of saidcompetitor and target nucleic acids.
 19. The method of claim 1 whereinat least one additional competitor nucleic acid is combined with saidtest nucleic acid sample in step (c), wherein said competitor nucleicacid is chosen such that it will generate an amplicon of a lengthdistinguishable from other amplicons generated in said amplification ofstep (d) and will be amplified by said first and second amplificationprimers with similar efficiency to the amplification of the otheramplicons generated in said amplification step.
 20. The method of claim19 wherein said additional competitor nucleic acid is added to said testnucleic acid sample at a known concentration that differs from theconcentration of said first or second competitor nucleic acids by atleast one order of magnitude.
 21. The method of claim 1 wherein a secondset of amplification primers specific for a second target nucleic acidand corresponding competitor nucleic acids of differing lengths and inknown concentrations are added, such that at least two target nucleicacids are quantitated in the same reaction by said amplification anddetection steps.
 22. The method of claim 21 wherein the target nucleicacid and second target nucleic acid are RNAs and wherein said methodmeasures gene expression of said target RNAs.
 23. The method of claim 1wherein said separating step is performed on a sample taken aftercompletion of said amplification.
 24. The method of claim 1 wherein saidseparating step comprises separation of nucleic acids in a plurality ofsamples removed from said amplification reaction during the course ofsaid amplification reaction.
 25. The method of claim 24 furthercomprising generating a profile of said amplification reaction based ondetection of said target amplicon during said amplification reaction.